The invention relates to the object that is characterized in the claims, i.e., a process for the production of gaseous microparticles for ultrasonic diagnosis, whose wall material is built up from polyesters of xcex1-, xcex2- or xcex3-hydroxycarboxylic acids, particles that can be produced according to this process, as well as their use in medical diagnosis.
Ultrasonic diagnosis has been used very extensively in medicine because of the trouble-free, simple handling. Ultrasonic waves are reflected at interfaces of different types of tissues. The echo signals that are produced in this case are electronically enhanced and made visible.
The visualization of blood vessels and internal organs using ultrasound generally does not allow the visualization of the blood flow that is present in it. Liquids, especially blood, yield ultrasonic contrast only if density and compressibility differences exist compared to the surrounding area. In medical ultrasonic diagnosis, e.g., substances that contain gases or that produce gas are used as contrast media since the impedance difference between gas and surrounding blood is considerably greater than that of liquids or solids and blood [Levine, R. A., J. Am. Coll. Cardiol. 3 (1989) 28; Machi, I. J. CU 11 (1983) 3].
Roelandt et al. [Ultrasound Med. Biol. 8 (1982) 471-492] describe that cardial echo contrasts can be achieved by peripheral injections of solutions that contain fine gas bubbles. These gas bubbles are created in physiologically compatible solutions by, e.g., shaking, other stirring or by addition of carbon dioxide. They are not standardized with respect to number or size, however, and can be reproduced only inadequately. Also, they are generally not stabilized, so that their service life is short. Their average diameters in most cases exceed that of an erythrocyte, so that it is not possible for them to pass through the pulmonary capillaries with subsequent contrasting of organs such as left heart, liver, kidney or spleen. Moreover, they are not suitable for quantification since the ultrasonic echo that they produce consists of several processes that cannot be separated from one another, such as bubble production, coalescence, and dissolution. Thus, it is not possible, e.g., with the aid of these ultrasonic contrast media to obtain information on transit times by measuring the history of the contrast in the myocardium. To this end, contrast media are needed whose scatter elements have sufficient stability.
EP 0 131 540 describes the stabilization of gas bubbles by sugar. This improves the reproducibility and homogeneity of the contrast effect, but these bubbles do not survive passing through the lungs.
EP 0 122 624 and 0 123 235 describe that the gas bubble-stabilizing effect of sugars, sugar alcohols, and salts is improved by the addition of surface-active substances. The passage through pulmonary capillaries and the possibility of visualizing the arterial femoral blood vessel and various organs such as the liver or spleen are provided for with these ultrasonic contrast media. In this case, however, the contrast effect is limited to the vascular lumen since the bubbles are not absorbed by the tissue cells.
None of the ultrasonic contrast media described remains unaltered in the body for a prolonged period of time. Organ visualization with sufficient signal intensity by selective concentration after i.v. administration or quantification is not possible with these agents.
Encapsulation of gases, such as, for example, air as ultrasonic contrast media is described in EP 0 224 934. The wall material that is used in this case consists of protein, especially human serum albumin with the known allergenic properties, which may also be accompanied by cytotoxic effects caused by denaturation.
Patent EP 0 327 490 describes gaseous microparticles for ultrasonic diagnosis based on biodegradable, synthetic materials. As biodegradable polymers, i.a., xcex1-, xcex2- or xcex3-hydroxycarboxylic acids are also disclosed. These agents have a sufficient in-vivo service life and, after intravenous administration, they concentrate intracellularly in the reticuloendothelial system and thus also in the liver or spleen, but the particles that are produced according to the examples of this application and that are based on hydroxycarboxylic acids have only a relatively small backscatter coefficient. The diagnostic action of contrast medium preparations that are prepared from them is therefore not satisfactory in all cases.
The object of this invention was therefore to provide an improved process for the production of microparticles based on polyesters of xcex1-, xcex2- or xcex3-hydroxycarboxylic acids, according to which it is possible to obtain particles that have a better backscatter coefficient than the particles of the prior art.
The particles and contrast medium preparations that are prepared from them should further meet the other requirements that are imposed on a modern contrast medium, such as, e.g., be small and stable enough to reach the left half of the heart after intravenous administration without significant gas loss and basically quantitative, to have good compatibility without having an allergenic potential, and not to agglomerate in an aqueous medium.
This object is achieved by this invention.
It has been found that microparticles based on polyesters can be produced from xcex1-, xcex2- or xcex3-hydroxycarboxylic acids, by the respective polyester and optionally a surface-active substance in an organic solvent or solvent mixture, of which at least one solvent is readily water-miscible, being dissolved, then a liquid perfluoro compound or water being dispersed in this solution, and then this dispersion being dispersed in water that contains a surface-active substance with the aid of a stirring mechanism, whereby the solvent is removed by pumping in gas and applying a vacuum. In this case, particles that contain first water or the liquid perfluoro compound precipitate out. Then, the suspension that contains particles is mixed with a suitable pharmaceutically acceptable cryoprotector and freeze-dried, whereby the liquid that is contained in the particles largely escapes, and is replaced after the freeze-drying device is aerated with the desired gas (generally air). Depending on the drying time, optionally a small amount of liquid (water or perfluoro compound) remains as vapor in the particles.
As a perfluoro compound, preferably perfluoropentane, perfluorohexane, perfluoro-1,3-dimethylcyclohexane, perfluorocyclohexene, perfluorodecalin, or perfluoroether is used.
Used as polymers in the process according to the invention are:
Polyglycolide (PGA), as well as its copolymers with L-lactide (PGA/PLLA) or polylactide (PLA), as well as its stereocopolymers such as, e.g., poly-L-lactide (PLLA), poly-DL-lactide or poly-L-lactide/DL-lactide or poly-xcex2-hydroxybutyrate (PHBA), as well as its copolymers with xcex2-hydroxyvalerate (PHBA/HVA) or poly-xcex2-hydroxypropionate (PHPA) or poly-p-dioxanone (PDS) or poly-xcex4-valerolactone or poly-"xgr"-caprolactone.
In the copolymers between lactic acid (LA) and glycolic acid (GA), the molar ratio relative to the monomers is in the range of 85:15 to 50:50, preferably 75:25.
As organic solvents or solvent mixtures for the polymers, dichloromethane, acetone, ethyl acetate, methyl acetate, triacetin, triethyl citrate, ethyl lactate, isopropyl acetate, propyl formate, butyl formate, ethyl formate, and/or methyl lactate are preferably used.
As a surface-active substance (surfactant), substances from the group of Poloxamers(R) or Poloxamines(R), polyethylene glycol alkyl ethers, polysorbate, saccharose esters (Sisterna The Netherlands), saccharose esters [Ryoto sugar esters, (Tokyo)], gelatin, polyvinylpyrrolidone, fatty alcohol polyglycoside, Chaps (Serva), Chap (Calbiochem), Chapso (Calbiochem), decyl-xcex2-D-glycopyranoside, decyl-xcex2-D-maltopyranoside, dodecyl-xcex2-D-maltopyranoside, sodium oleate, polyethylene glycol, polyvinyl alcohol or mixtures thereof are used.
As gases, air, nitrogen, oxygen, noble gases, dinitrogen oxide, carbon dioxide, halogenated hydrocarbons, saturated or unsaturated hydrocarbons in gaseous form, nitrogen dioxide, and/or ammonia are used.
An alternative process for the production of microparticles based on polyesters of xcex1-, xcex2- or xcex3-hydroxycarboxylic acids consists in the desired polymer(s) and optionally an amino acid being dissolved in at least one organic solvent and this solution being sprayed via a nozzle into a column that is filled with a supercritical gas or through which the latter flows, whereby the solvent is taken up by the supercritical gas.
As polymers or copolymers, the same substances as were mentioned in the first-mentioned production process are used in this process variant. The equivalent applies for the mixing ratios in the copolymers. Preferably, an amino acid is provided instead of the surfactant in the dissolved polymers (polyesters) that are used in each case. As amino acids, preferably L-lysine, L-phenylalanine, L-tryptophan, as well as D,L-phenylalanine are used.
As solvents or solvent mixtures, the solvents that are cited in the first process variant are suitable.
The addition of a perfluoro compound is not necessary in this case.
As supercritical gases, dinitrogen oxide, carbon dioxide, halogenated hydrocarbons, saturated or unsaturated hydrocarbons, nitrogen dioxide, and/or ammonia are used, wherein carbon dioxide is preferred. The supercritical gases can optionally contain up to 10% additives such as, e.g., lower alcohols, such as, e.g., ethanols, esters, or gases, such as, e.g., nitrogen.
The size of the resulting particles can be controlled by type, size and shape of the injection nozzle, working pressure and temperature in the column. A particle size as is necessary for an intravenously administered ultrasonic contrast medium ( less than 10 xcexcm) can be obtained by using a nozzle with a nozzle diameter of 0.5 mm and a spraying angle of 10xc2x0 at a working pressure of between 90 and 100 bar, preferably 94-96 bar and a temperature of 36xc2x0 C.
Another aspect of the invention relates to microparticles for ultrasonic diagnosis that can be produced according to the above-named process.
Another aspect of the invention relates to contrast media for ultrasonic diagnosis that contain microparticles, which were produced according to the above-mentioned process.
These agents can be obtained by the dried microparticles being resuspended in a pharmaceutically acceptable suspension medium.
As pharmaceutically acceptable suspension media, for example, water p.i., aqueous solutions of one or more inorganic salts, such as physiological electrolyte solutions and buffer solutions, such as, e.g., Tyrode""s solution, aqueous solutions of mono- or disaccharides such as glucose or lactose, sugar alcohols such as mannitol, which optionally in addition have a surface-active substance, e.g., from the group of polysorbates or polysaccharides, polyvinylpyrrolidone, polyethylene glycol, saccharose mono- and diesters or substances from the group of poloxamers or poloxamines or their mixtures and/or a physiologically compatible multivalent alcohol such as glycerine are suitable. Preferred is, however, water that is suitable for injection purposes.
To increase the reliability of administration, the suspension can be filtered immediately before injection.
The microparticles that are produced according to the process of the invention that are based on polyesters of xcex1-, xcex2- or xcex3-hydroxycarboxylic acids or contrast media that are prepared from them meet all requirements that are set on an ultrasonic contrast medium. The particles that are contained in the agents are distinguished by the following advantages:
They are quickly degraded in vivo,
degradation products are toxicologically harmless,
they circulate for a sufficiently long period in the blood circulation,
they can be used in all modes of ultrasonic diagnosis, especially also in modes in which nonlinear effects are used,
they are well-tolerated,
they have a uniform, controllable size distribution,
they are easy to produce,
the polymeric particles have a narrow molecular weight distribution,
they are sufficiently stable to survive passing through the lungs and thus are also suitable for contrasting the left heart, and
they are taken up by the reticuloendothelial system and thus are also suitable for contrasting the liver and spleen.
The particles that are produced according to the process of the invention especially have a larger backscatter coefficient than the particles that are disclosed in EP 0 327 490 which also are built up from xcex1-, xcex2-, xcex3-hydroxycarboxylic acids. This was also shown in a comparison test. Thus, for a freshly prepared suspension of the particles in water described in EP 0 327 490 (Example 1) (concentration 18.6 mg/ml) after 5 minutes, a backscatter coefficient of xcex1s=4xc3x9710xe2x88x924 [Db/cm] at 5 MHz of transmitter frequency was measured, while for a preparation according to the invention (particles according to Example 1 suspended in water/concentration 0.18 mg/ml) under otherwise identical conditions, a backscatter coefficient of as xcex1s=2.7xc3x9710xe2x88x921 [dB/cm] was measured. It is taken into consideration that the concentration of the preparations according to the invention was smaller by a factor 100 than the comparison preparation, i.e., an increase of the backscatter coefficients by five orders of magnitude results. Since the imaging effect depends directly on the backscatter coefficient, the particles that are produced according to the invention represent considerably more effective contrast media.
The determination of the backscatter coefficient is done in an in-vivo experimental set-up, in which the backscattered signal caused by a contrast medium that is present in a vessel is measured (see also xe2x80x9cStandardization of the Measurement of Acoustical Parameters of Ultrasound Contrast Agentsxe2x80x9d First European Symposium on Ultrasound Contrast Imaging, Jan. 25-26, 1996, Rotterdam).
The examples below are used for a more detailed explanation of the object of the invention, without intending that it be limited to these examples.